Glioblastoma (GBM) is a highly aggressive and malignant brain tumor, characterized by hypoxia in its microenvironment, which drives its growth and resistance to treatments. Hypoxia-inducible factor 1 (HIF-1) plays a central role in GBM progression by regulating cellular adaptation to low oxygen availability, promoting processes such as angiogenesis and cell invasion. However, studying and modeling GBM under hypoxic conditions is complex, especially due to the limitations of animal models. In this study, we developed a glioma spheroid model using an alginate–gelatin hydrogel scaffold, which enabled the simulation of hypoxic conditions within the tumor. The scaffold-based model demonstrated high reproducibility, facilitating the analysis of HIF-1α expression, a key protein in the hypoxic response of GBM. Furthermore, cell viability, the microstructural features of the encapsulated spheroids, and the water absorption rate of the hydrogel were assessed. Our findings validate the three-dimensional (3D) glioblastoma spheroids model as a valuable platform for studying hypoxia in GBM and evaluating new therapies. This approach could offer a more accessible and specific alternative for studying the tumor microenvironment and therapeutic resistance in GBM.

Glioblastoma is the most prevalent and fatal form of primary brain tumors. New targeted therapeutic strategies for this type of tumor are imperative given the dire prognosis for glioblastoma patients and the poor results of current multimodal therapy. Previously reported drawbacks of antibody-based therapeutics include the inability to translocate across the blood–brain barrier and reach intracellular targets due to their molecular weight. These disadvantages translate into poor target neutralization and cancer maintenance. Unlike conventional antibodies, vNARs can permeate tissues and recognize conformational or cryptic epitopes due to their stability, CDR3 amino acid sequence, and smaller molecular weight. Thus, vNARs represent a potential antibody format to use as intrabodies or soluble immunocarriers. This review comprehensively summarizes key intracellular pathways in glioblastoma cells that induce proliferation, progression, and cancer survival to determine a new potential targeted glioblastoma therapy based on previously reported vNARs. The results seek to support the next application of vNARs as single-domain antibody drug-conjugated therapies, which could overcome the disadvantages of conventional monoclonal antibodies and provide an innovative approach for glioblastoma treatment.

Glioblastoma is the most common and highly malignant brain tumor in adults. New targeted therapeutic approaches are imperative. EGFRvIII has appealing therapeutic targets using monoclonal antibodies. Thus, endeavors toward developing new mAbs therapies for GBM capable of targeting the tumor EGFRvIII biomarker must prevail to improve the patient’s prognosis. Here, we isolated and characterized an anti-EGFRvIII vNAR from a non-immune freshwater stingray mixed library, termed vNAR R426. The vNAR R426 and pEGFRvIII interaction was demonstrated by molecular docking and molecular dynamics, and the recognition of EGFRvIII in vitro was further confirmed by cell immunofluorescence staining. Moreover, the vNAR R426 was shown to be an effective cisplatin drug carrier in the U87-MG glioma cell line. The cisplatin-coupled vNAR demonstrated highly significant differences when compared to free CDDP at 72 h. Notably, the cisplatin-vNAR carrier achieved better efficacy in the U87-MG cell line. Thus, we described the vNAR R426 internalization by receptor-mediated endocytosis and the subsequent COPI-mediated nuclear translocation of EGFRvIII and highlighted the importance of this shuttle mechanism to enhance the targeted delivery of cisplatin within the glioma cell’s nucleus and improved cytotoxic effect. In conclusion, vNAR R426 could be a potential therapeutic carrier for EGFRvIII-targeted glioblastoma and cancer therapies.